DESCRIPTION (applicant's abstract): The membranes of mammalian cells are composed of an array of phospholipid species that are now recognized to function as a diverse source of lipid mediators. These mediators function as both intercellular and intracellular signals and are key components of numerous signaling cascades. The levels of ether-linked phospholipids vary greatly among cells; but, in a number of cells they are known to serve as an important reservoir of arachidonic acid and as a precursor of platelet activating factor (PAF), one of the most active mediators known. The ether bonds are not hydrolyzed by phospholipases, and distinct pathways are required for their synthesis and turnover. We recently completed an analysis of the subclass composition of the choline- and ethanolamine-containing phosphoglycerides (PC and PE) of four human breast tumor cell lines and found that the two cell lines that are estrogen receptor-positive (ER+), MCF-7 and T47D, contain only traces of alkyl or alk-1-enyl (plasmalogens) species in PC and similarly low levels in FE. In contrast the estrogen receptor-negative (ER-) cell lines, MDA 231 and MDA 435, contain 22 and 13 mol percent respectively of the alkyl subclass in PC and >30 mol percent plasmologen in the PE. Primary breast tumors contained higher ether content than the surrounding normal tissues. We have shown that the ER- MDA 231 cells can synthesize PAF. Breast tumor cells are known to contain phospholipase D that can be activated to convert PC to phosphatidic acid and diglycerides. PAF is known to promote cell growth and angiogenesis, whereas lyso PA promotes cell growth and tumor cell invasiveness; PA is believed to activate Raf1 and Ras. It is our hypothesis that the more invasive and metastatic ER- cells will produce alkyl-linked species of these mediators, along with PAF, and that they will be more persistent and potent mediators than their ester-linked counterparts and thus contribute to the aggressiveness of the ER- cells. Our specific aims are: (1) to measure the production of ether-linked mediators in estrogen receptor-positive and -negative breast tumor cells and compare our findings in more normal MCF I OA cells; (2) to determine the pathways by which alkyl acyl-GPC is converted to products in the breast tumor cells and to elucidate the mechanisms by which the pathways are controlled; and (3) to determine the action of ether-linked mediators on key signaling pathways of the estrogen receptor-positive and estrogen receptor-negative breast tumor cells and to measure their effect on cell growth. The proposed studies promise to advance our understanding of the role of the ether-linked lipids in tumor cells at the molecular level and could lead to new therapeutic approaches and targets.